1 2 bis 2 aminophenoxy ethane n n n n tetraacetic acid tetrakis acetoxymethyl ester bapta am

Human lung fibroblast cell line MRC5 cells were purchased from American Type Culture Collection (Rockville, MD). Fura-2-AM was purchased from Teflabs (Austin, TX). U73122 and its inactive analog, U73343 were from Tocris (Minneapolis, MN). Caffeine, clotrimazole (CLZ), 3-Aminobenzamide (3-AB), N-(p-amylcinnamoyl) anthranilic acid (ACA), 2-aminoethoxydiphenyl borate (2-APB), Chlorpromazine (CLP), N-acetylcysteine (NAC), lanthanum chloride (LaCl₃), and all other chemicals were from Sigma. Dulbecco's Modified Eagle's Medium (DMEM), penicillin-streptomycin, trypsin-ethylenediaminetetraacetic acid (EDTA), 5-(and-6)-choloromethyl-2′,7′-dicholorodihydrofluorescin diacetate (CM-H2DCFDA), phosphate-buffered saline (PBS), fetal bovine serum (FBS), Pluronic F-127 (20% in DMSO), and 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis, acetoxymethyl ester (BAPTA,AM) were from Invitrogen (Carlsbad, CA). MRC5 cells were incubated at 37℃ in a humidified 5% CO₂/95% air atmosphere in DMEM containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. When the cell culture reached 80% confluence, cells were dispersed by adding trypsin-EDTA for 2 min and then transferred to new culture dishes or to glass coverslip-covered dishes for Ca²⁺ measurements.

Pregnanolone (3α‐hydroxy‐5β‐pregnan‐20‐one) was from Sigma. Pregnanolone sulfate (20‐oxo‐5β‐pregnan‐3α‐yl 3‐sulfate) and pregnenolone sulfate (20‐oxo‐pregn‐5‐en‐3β‐yl 3‐sulfate) were prepared as pyridinium salts, as described previously (Stastna et al., 2009 (link), for pregnanolone sulfate; Krausova et al., 2018 (link), for pregnenolone sulfate; method based on Arnostova et al., 1992 ). Bicuculline methochloride, TTX, NBQX, phorbol 12,13‐dibutyrate (PDBu), thapsigargin, bisindolylmaleimide I (GF109203X) and calphostin C were obtained from Tocris. 1,2‐Bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA‐AM) was purchased from Invitrogen. Reagents for the preparation of extracellular and intracellular solution, and glutamate, glycine, strychnine and ionomycin were from Sigma or AnalaR. Cell culture media and solutions were from Gibco/Invitrogen and Serva. Molecular biology reagents were from Biolife and Geneaid.